Glutathione Beads 4FF By Lablead biotech,

La description

CATï¼šG****0

Price: $**- 5ml; $****5ml; $**4—*0ml;$**1—**0ml;$***4—**0ml (negotiable)

Storageï¼š**8°C

product specificationï¼š
Glutathione Beads 4FF allows the purification of glutathione-S-transferase, glutathione-dependent proteins, and recombinant derivatives of glutathione transferase expressed by various expression systems in one step. Glutathione Beads 4FF is made from a highly cross-linked 4% agarose gel based on a **-atom spacer arm that chemically covalently binds reduced glutathione. Glutathione Beads 4FF is suitable for industrial large-scale protein purification due to its pressure-resistant substrate, which enables the purification of target proteins at relatively high flow rates.
Chart 1 Glutathione Beads 4FF feature

Item	Feature
Matrix	Highly crosslinked 4% agarose gel
ligand	Glutathione coupled by a **-atom spacer arm
capacity	>0 mg GST-tagged protein(0 kDa) /ml resin
Microsphere size	*****5 um
maximum pressure	0.3 MPa, 3 bar
pH stability range	***2
Storage buffer	1xPBS containing *0% ethanol
storage temperature	**8°C

Purification process
1. Buffer preparation
The buffer is best filtered with 0.*2um or 0.*5um filter before use.
Balance/wash solution: **0 mM NaCI, 2.7 mM KCI, *0 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4
Eluent: Preparation of *0mM reduced glutathione with equilibrium solution (for immediate use)
Note: ***0 mM DTT can be added to the equilibrium solution and eluent
2. Sample preparation
Samples are recommended to be centrifuged or filtered with 0.*2um or 0.*5um filter membrane before loading to reduce impurities, improve protein purification efficiency and prevent clogging of columns.
2.1 protein expressed by bacteria or yeast
1) Select a single colony into the medium, and add the corresponding concentration of inducer to induce the corresponding time according to the carrier instructions.
2) After the expression, the culture medium was transferred to a centrifuge cup at ***0 rpm (***0xg), centrifuge for *5 min, collect the bacteria, and then add the equilibrium liquid according to the bacteria: equilibrium liquid = 1:*0 (W/V), and add PMSF with a final concentration of 1 mM. Add lysozyme (working concentration is 0.**0.4 mg/ml, if the expressed host cell contains pLysS or pLysE, you can not add lysozyme, while you can also add other protease inhibitors, but can not affect the binding of the target protein to the filler).
3) Suspend the bacterial sediment (if the concentration of bacterial solution is high, you can also consider adding *0 ug/ml RNase A and 5 ug/ml DNase I), mix it well, place it on ice, and then ultrasonic break the cells on ice until the bacterial solution is basically clarified.
4ï¼‰Transfer the clarified crushing liquid to a centrifuge tube at ****0 rpm (****0xg) and centrifuge at 4°C for ****0 min. Remove supernatant and set aside on ice or store at **0°C.

2.2 Yeast, insect and mammalian cells secrete and express soluble proteins
1) Transfer the cell culture medium to a centrifuge cup, ***0 rpm (***0xg), centrifuge for *0 min, collect the bacteria and obtain the supernatant, which can be directly added to the column for use.
2) For a large volume of supernatant, it is necessary to add ammonium sulfate to precipitate and concentrate, and the protein can be added to the column after dialysis with the equilibrium solution.

3. Loading of chromatographic column
3.1 Loading of gravity column
1) Take a gravity chromatographic column of appropriate specifications, load it into the lower gasket, add an appropriate amount of pure water washing column tube and gasket, and close the lower outlet.
2) Mix Glutathione Beads 4FF evenly, absorb appropriate amount of slurry with the gun head and add it into the gravity column (the actual volume of the medium accounts for half of the suspension), and open the lower outlet to drain the protective liquid.
3) Add appropriate amount of pure water to wash the medium, and close the lower outlet after the liquid gravity flow in the column tube dries.
4) Load the upper gasket after wetting, ensure that there is no gap between the gasket and the filler, and maintain the level.
5) The loaded gravity column can be directly added to the balance liquid for balance, and the protective liquid is added when not in use for the time being, and stored at **8°C.

3.2 Loading of medium pressure chromatographic column
Before column installation, the column bottom area is calculated according to the diameter of the chromatographic column, and the required medium volume is calculated according to the required column height. The formula is as follows:
V = 1.*5πr2h (V: required medium volume ml; 1.*5: compression factor; r: column tube radius cm; h: Filling height cm)
Note: The volume of suspended fluid taken should be twice the volume of the medium, because the medium volume only accounts for half of the total volume of the suspension, and the other half is the protective fluid.
1) Rinse the sieve plate and joint at the bottom of the chromatography column with deionized water to ensure that there are no bubbles on the sieve plate at the bottom of the column, close the outlet at the bottom of the column, and leave **2 cm of deionized water at the bottom of the column.

2) Suspend the medium and carefully pour the slurry continuously into the chromatographic column. A glass rod is used to pour slurry along the wall of the column to reduce the formation of air bubbles.
3) If a reservoir is used, fill the chromatographic column and reservoir with water immediately, place the sample dispenser on the surface of the slurry, and connect it to the pump to avoid bubbles in the dispenser or sample tube.
4) Open the bottom outlet of the chromatographic column, turn on the pump, and make it carry out at the set flow rate. The buffer should be allowed to flow slowly through the chromatographic column initially and then slowly increased to the final flow rate, so as to avoid hydraulic impact on the formed column bed and avoid uneven formation of the column bed. If the recommended pressure or flow rate is not reached, the maximum flow rate of the pump used can be used, which can also get a good filling effect. (Note: In subsequent chromatographic procedures, do not exceed *5% of the maximum column flow rate.) When the column bed height is stabilized, add at least 3 times the column bed volume of deionized water at the final column flow rate. Mark the height of the post bed.
5) Turn off the pump and close the outlet of the chromatographic column.
6) If using a reservoir, remove the reservoir and place the dispenser in the chromatographic column.
7) Push the dispenser towards the column to the marked column bed height. Allow the loading fluid to enter the distributor and lock the distributor connector.
8) Connect the loaded chromatographic column to the pump or chromatographic system and start balancing. Re-adjust the distributor if necessary.

4. Sample purification process
4.1 Purification by incubation
1) Glutathione Beads 4FF was added into the centrifuge tube according to the purified sample quantity, centrifuged at ***0 RPM for 1 min, and then the supernatant was absorbed and discarded. It can also be added to the gravity column to drain the protective fluid.
2) Add 5 times the volume of the medium balance liquid cleaning medium into the centrifugal tube, centrifuge at ***0rpm for 1 min, and discard the supernatant; If the gravity column is used, it is cleaned directly in the gravity column and the balance liquid is dried directly by gravity flow; Repeat more than twice.
3) Add the sample, close the centrifugal tube or gravity column tube, and incubate at 4°C for **4h or *7°C for *0 min*2h.
4) After incubation, centrifuge at ***0 rpm for 1 min, absorb and discard the supernatant, or filter the collection medium, and retain the supernatant as a stream for electrophoresis identification.
5) Clean the medium with a detergent of 5 times the volume of the medium, centrifuge at ***0 rpm for 1 min or filter with a gravity column tube, remove the supernatant (be careful not to suck the medium), repeat **5 times, and it is recommended to replace a new centrifugal tube in the middle.
6) Add **5 times column volume of eluent for elution, incubate at room temperature for ****5 min, centrifuge at ***0 rpm for 1 min or collect eluent in gravity column tube, which can be repeated **3 times.

4.2 Purification by gravity column method
1) The filled Glutathione Beads 4FF gravity column is balanced with 5 times the column volume balancing liquid, so that the filler is placed under the same buffer liquid system as the target protein, and this is repeated **3 times.
2) Add the sample to the balanced gravity column, the sample retention time is at least 2 min, ensure that the sample and the medium are in full contact, collect the effluent, and the sample can be repeatedly loaded to increase the binding efficiency.
3) Use ****5 times the column volume of the detergent to wash the detergent, remove the impurity protein unless specifically adsorbed, and collect the detergent.
4) Eluent of ***0 times column volume is used, collected in stages, one tube is collected for each column volume, and tested separately, which can ensure that all bound target proteins are eluded, and high purity and high concentration of proteins can be obtained.
4.3 Purification by medium pressure chromatography column
Once the Glutathione Beads 4FF is filled, it can be used with a variety of conventional low and medium pressure chromatography systems.
1) Fill the pump pipe with deionized water. Remove the upper plug, connect the chromatographic column to the chromatographic system, open the lower outlet, connect the preassembled column to the chromatographic system, and tighten.
2) Flush out the storage buffer with **5 times the column volume of deionized water.
3) Balance the column with a balancing liquid that is at least 5 times the column bed volume.
4) Use pump or sample ring to sample.
Note: The increased viscosity of the sample results in a large backpressure of the chromatographic column, even if the sample volume is small. Do not exceed the binding capacity of the column. The large sample volume can also cause a lot of back pressure, making the injector more difficult to use.
5) Wash the column with detergent until UV absorption reaches a stable baseline (usually at least ****5 column volumes).
6) Eluent of ***0 times column volume is used to collect eluent, that is, the target protein component.
After the elution of the above steps, rinse 3 times the column volume with the equilibrium solution, then rinse 5 times the column volume with pure water, and then rinse 2 column volumes with the protective solution, and then store the medium at **8°C.

5. SDS-PAGE examining
Samples obtained from purified products (including effluent, washing and elution components) as well as original samples will be tested for purification using SDS-PAGE.

6. Packing cleaning
GST label protein purification products can be reused without regeneration, but with the increase of non-specific binding proteins and protein aggregation, often resulting in decreased flow rate and binding load performance, at which time the filler needs to be cleaned

To remove some precipitated or denatured substances, the following methods are recommended
Clean with 2 times the column volume of 6 M guanidine hydrochloride solution, then immediately clean with 5 times the column volume of PBS, pH 7.4.
Remove some non-specific adsorbent substances caused by hydrophobic adsorption
Clean with **4 times the column volume *0% ethanol or 2 times the column volume 1% Triton X***0, then immediately clean with 5 times the column volume PBS, pH 7.4.

Pays:	China
N Â° de modÃ¨le:	-
Prix FOB:	(Negotiable) Obtenir le dernier prix
LocalitÃ©:	China
Prix de commande minimale:	-
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Packaging Detail:	-
Heure de livraison:	7-15 days if in stock
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Groupe de produits :	Protein Biology